Massively parallel sequencing and next generation sequencing platforms are rapidly transforming data collection and analysis in genome, epigenome and transcriptome research. Most of these sequencing technologies read relatively short oligonucleotide sequences from the ends of DNA fragments. For examples, sequencers from Illumina and Applied Biosystems provide reliable reads up to about 50 bases, and Paired-End-Tag (PET) method reads only 20 or 27 bases. This is due to the DNA processing enzymes MmeI or EcoP151 used to generate the tags.
Efficiency of ChIP assay is generally low with only two copies of targets per cell usually possible. Transcription factor binding may not be stable at such low values. 1%=˜20 molecules/locus in final IP sample.
Degenerate Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) is a robust method to amplify trace amount of DNA for various downstream applications such as sequencing and genotyping. However, this method relies on addition of sequences (>18 bases) to the ends of original DNA fragments—making it unfavorable for next generation sequencing as they produce only short sequence reads. DOP-PCR utilise 3′ degenerate sequence part of primers for initial library synthesis and fixed 5′ sequence for following exponential amplification by PCR (see FIG. 8). DOP-PCR has several problems:                (i) Fixed 5′ primer sequence at the ends of amplified fragments. Every amplification product has primer sequence: at both ends as a result of PCR amplification.        (ii) Mutations introduced by degenerate priming and PCR amplification. Non-perfect annealing of degenerate oligo primers during library synthesis and mispriming events during PCR cause mutation and addition of sequences at the ends of amplified fragments.        